The Role of Phosphodiesterases in Cyclic Nucleotide Compartmentation Across Different Pathways in the Adult Rat Ventricular Myocyte

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In cardiac myocytes, multiple receptor mediated signalling pathways converge on cyclic nucleotide production. These second messengers act to achieve changes in cellular function. Despite this, each signalling molecule and receptor can achieve distinct sub-cellular effects. This has led to the theory of cyclic nucleotide compartmentation, which has been postulated to be mediated by phosphodiesterases (PDEs). Research in this field has focused on compartmentation using β-adrenergic stimulation. As an extension of this work, we investigated the effects of two agonists, prostaglandin E2 (PGE2; 10 nM) and forskolin (FSK; 30 nM), on various cellular parameters in the presence of either cilostamide (1 µM) a selective PDE3 inhibitor, or Ro 20-1724 (10 µM) a selective PDE4 inhibitor. In myocytes treated with PGE2, unloaded cell shortening and intracellular calcium transients exhibited significantly different (p<0.05) values of 147 ± 10% and 138 ± 5% of pre-treatment (t=0) values, respectively, in the presence of PGE2 and Ro 20-1724 (all n=5). However, values were not significantly different in cells pre-treated with cilostamide. Conversely, FSK resulted in significant increases of 153 ± 9% (n=5; P>0.05) and 189 ± 20% (n=5; P>0.05) of t=0 in cells treated with cilostamide and Ro 20-1724, respectively. PGE2 enhanced ICa,L was not altered using either PDE inhibitor. However, with FSK as an agonist, a significant increase in peak ICa,L from -6.0 ± 0.8 pA/pF to -7.7 ± 0.4 pA/pF (n=5; P>0.05) was observed in cells pre-treated with Ro 20-1724. SR calcium loading was also increased, but only in cells pre-treated with Ro 20-1724, with values of 127 ± 11% and 156 ± 47% of t=0 (n=5) for FSK and PGE2, respectively. Our results demonstrate that a unique pattern of regulation exists for PGE2 and that it is different from what was found previously with isoproterenol. We have shown that this is achieved by functionally localizing PDEs to distinct compartments. Specifically, PDE4 is localized at the SR, PDE3 at the sarcomere, and a combination of both at the calcium channel. However, our ICa,L results also indicate that the location of the receptor and adenylate cyclases must be considered relevant to compartmentalizing the cAMP signal.